Harnessing the Blinking Heterogeneity of Fluorescent Probes: A New Take on Single-Molecule Research

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In this project, funded by the Chemical Structure, Dynamics & Mechanism B Program and the Chemical Measurement and Imaging Program of the Chemistry Division, Kristin L. Wustholz of the Department of Chemistry at College of William and Mary will examine how light is emitted from molecules in order to enhance the ability to visualize structures at the nanoscale. When molecules are continuously illuminated, they respond by emitting random bursts of light, a phenomenon called blinking. This blinking behavior can be harnessed for many applications such as imaging biological structures and nanomaterials down to 10-nm length scales – a feat that is not possible with conventional optical microscopy. However, lack of understanding and control over the variations in blinking behavior from molecule to molecule across a sample fundamentally limit the image quality that can be achieved. The focus of this project is to examine and engineer blinking to produce high-quality and super-resolution images of materials that are relevant to medicine and technology. At the same time, this project provides training for undergraduate and master’s research students who learn physical, analytical, and computational techniques as well as build communication skills. Dr. Wustholz will continue her commitment to diversity and inclusion through mentoring with the Chemistry Women Mentorship Network and her use of teaching practices that benefit all students, but especially those who are low-income, first-generation, and underrepresented. This project also involves a new collaborative exchange with Temple University to further enhance scientific training and professional development of students at both institutions.The design and development of improved single-molecule probes is currently limited by several factors including: 1) unwanted heterogeneity in blinking dynamics due to contributions from multiple dark states, 2) uncontrolled environment-induced changes to blinking, and 3) reliance on spectrally-distinct dyes for multicolor imaging. The first aim of this project is to untangle the role of different dark states and their dependence on molecular structure so that blinking heterogeneity can be minimized when it is detrimental and exploited when it is useful. The initial target is a series of well-characterized xanthenes as a model system to unravel and control heterogeneity using a dark-state competition strategy. Next, the influence of molecular structure and environmental conditions will be used to control the different dark states and blinking heterogeneity of BODIPYs, promising probes whose blinking mechanisms are currently not well understood. These investigations will inform molecular design strategies for tailored emission dynamics as well as unlock new opportunities in probe design through the development of a new approach to multicolor super-resolved imaging.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.